Method and apparatus for counting microorganisms



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UnitedStates Patent O METHOD AND APPARATUS FOR COUNTING MICROORGANISMSThomas L. Snyder, Frederick, Md., assignor to the United States ofAmerica as represented by the Secretary of the Army ApplicationSeptember 17, 1953, Serial No. 380,888

2 Claims. (Cl. 195'-103.5)

(Granted under Title 35, U. S. Code (1952), sec. 266) The inventiondescribed herein may be manufactured and used by or for the Governmentof the United States of America without the payment to me of any royaltythereon.

This invention relates to improvements in method and apparatus forcounting microorganisms and in particular is concerned with means forthe determination of the number of viable biotics in a known volume ofHuid or carrier.

In the past it has been a serious problem in the field lof Bacteriologyto ascertain the concentration and nurnlb'er of microorganisms in `acarrier and there have been required complicated and pains-takingefforts in such de- One of the conventional processs comprisesincubating a culture on a Petri dish and then counting individually thenumber of microorganism colonies in the growth. Obviously this procedureis time consuming, laborious and leads to inaccuracies caused b3 theditliculty in counting. Other means for finding the concentration ofmicroorganisms have been devised which include pipetting small knownamounts of the culture into a plurality of nutrient-filled test tubesand after an incubation period counting the number of cloudy tubes andcorrelating this number to the concentration of viable organisms in thecarrier. This method is however quite cumbersomein that a large space isrequired for the test tubes and in carrying out the incubation stagewhile the `filling of the individual test tubes to the same level inobtaining equal volumes is another requirement necessitating specialmanipulations'and additional labor." i

By Ymeans of this invention the-complicated procedures and bulkyequipment required have been eliminated. The process and apparatus ofthis invention include ascertaining the number of microorganisms in anutrient liquid carrier by immersing a perforated stick in the liquid sothat the perforations or openings which are of capillary size arecharged with the liquid. The material used in this stick must be of anon-wetting characteristic in order that there be no film of liquidbetween the perforations.

t That is, the stick must be composed of material which is sullicientlywettable to exhibit capillarity characteristics while also possessingsuicient non-wettability so as to prevent the formation of any lm on thehat surface adjacent to these openings. This is of critical significancesince if the material is wet with only a lm of liquid of molecularthickness on the surface between the perforations, the microorganismswill be transmitted from one aperture to another using this thinlywetted surface as the conductor or migration medium. This inventioncontemplates broadly the use of halogenated hydrocarbons such as Teon,made by the E. I. Pont de Nemours Co., Inc., which has been found topossess the desirable nonwetting characteristics as well as beingchemically inert to the mediums used. The uniformly perforated capillaryopenings make possible the inclusion of the same volume of liquid withineach opening due to the capillary action which causes these openingsautomatically to be lled to Plce the same level. Obviously this obviatesany special procedures in the illing stage and greatly simplifies theoperation. Once the stick has been charged with the microorganism theincubation period is commenced at the end of which the determination ismade by counting the number of turbid cells indicating the growth ofcolonies. The perforations in the stick may be so arranged that acompletely automatic counting process can be used by photo-electricmeans and like scanning processes.

Accordingly, it is an object of this invention to provide a method andapparatus for simply and quickly determining the number ofmicroorganisms in a nutrient liquid carrier.

It is a further object of this invention to provide a method andapparatus for taking 4a multiplicity of samples from a liquid carrierwhich samples are of the same volume with only one piece of apparatusand in only one operation.

It is still another object of this invention to provide a process andapparatus for determining the concentration of microorganisms in anutrient liquid carrier by using a perforated relatively non-wettablecharge stick and subsequently detecting growth of the progeny in theperforations.

`Yet another object of this invention is to provide an inert perforatedliquid charge stick of relatively nonwettable characteristics which canbe used in a process for determining the number of microorganisms in anutrient liquid carrier so that the growth in the perforations can bereadily ascertained by automatic means.

`Other objects will be apparent from the description which follows andwill be further apparent to those skilled in the art.

ln the drawings:

Fig. l is a plan view of one form of the charge stick.

Fig. 2 is a cross-sectional view of Fig. l.

Fig. 3 is a plan view of a modified form of the charge stick.

Fig 4 is a cross-sectional view of Fig. 3.

Fig. 5 is a graph showing a comparison betweenresults obtained by thisinvention and the standard plate count.

Referring now to the drawings andFigs. 1 and 2A in vp'ir'ticular, it'will be. seen that the charge stickh is ,of` a'planiform nature and`has 'a' limited depth with perforations 11 of a capillary sizeextending therethrough. These perforations, as an example, may beuniformly distributed so that there are 2000 such holes in a 6% by 1%inch area. It has been found that such openings may conveniently have adiameter of 0.025 inch although it is to be understood that there is avariable range depending upon the viscosity of the fluid being handledand upon other factors. Likewise other spacings may be utilized. Thischarge stick may conveniently be used in automatic counting procedureswhere the individual holes are scanned by photo-electric cells and thelike with the turbidity resulting from growth of the progeny registeringas a growth cell whereas the sterile cells remain clear.

The depth (or thickness) of the stick is governed by the amount ofturbidity (length of perforation) it is desired to offer the detectinginstrument. The stick is always completely immersed in the carrierliquid and the holes completely filled, yagitation being employed ifnecessary. The stick is withdrawn from the liquid with its plane keptperpendicular to the surface of the liquid, in order to retain a uniformand maximum quantity of liquid in the perforations.

The modification shown in Fig. 3 at 13 is of the same generalconstruction as the charge stick disclosed in Figs. 1 and 2 with theexception that the spacing of the perfor.

ations is broken up so that the perforations are in individual banks 12with four of these banks across the widthl and ten along the lengthcomprising forty banks in all, with ifty perforations in each bank. Thisarrangement facilitates visual counting by the naked eye so that eachbank may be examined separately.

It is necessary that the material used in the construction of the chargesticks be absolutely inert to the medium and be of a non-wettablenature. The tetra-iluoroorganic resins have been found to be veryadvantageous in this respect and the material Teflon is used in thisconstruction. This resin is synthetic polymerized tetrauoro ethylenewhich has the desired non-wettable nature. Other plastics, such as theacrylic resin Lucite, have been tested and found to be wanting in thiswetting characteristic since they have formed thereon a thin molecularfilm on the surface of the charge stick between the perforations whichis ruinous for the successful operation of this process. Therequirements of this non-wetting characteristic are critical to thisinvention since as has been pointed out above even a molecular film willserve to facilitate the migration of the microorganisms.

In use:

A suitable nutrient suspension or other medium containing microorganismswhose concentration and number is to be determined is furnished and thecharge stick is immersed therein. The openings in the charge stick whichare of a uniform capillary size will draw therein a constant volume ofiiuid which is dependent on the viscosity of the fluid. The charge stickafter this charging operation can then be withdrawn and handled ineither a vertical or horizontal position since the liquid is retained inthe openings by capillary attraction.

The charge stickjcontaining the liquid to be analyzed is then set asidein a saturated aqueous atmosphere for a suitable incubation period afterwhich growth of the progeny in each opening is determined byascertaining whether or not it has turned cloudy or otherwise changed inform. The number of openings so changing may then be correlated to theconcentration and number of microorganisms in the medium in which thecharge stick Was was immersed by conventional formulation since thevolume of this medium and that of the liquid in the charge stick areascertainable.

Comparisons have been made between the standard plate counting methodand the process of this invention using the charge stick hereindisclosed on fourteen bacterial suspensions of Serrata marcescens. Theresults are illustrated by the graph of Fis- 5 which Shows the method ofthis invention gave higher values than the standard plate count. It isbelieved that these higher estimates represent more accurate counts ofthe viable organisms actually present because: (1) non-linearity of thecurve tends to eliminate an explanation of the discrepancy based onerror of calibration of the device, (2) preliminary statistical studiesdo not support the hypothesis of signilicant dispersion of growth frompositive holes to adjacent sterile holes, and (3) the standard platecount tends to be depressed at high counts per plate as a result ofcrowding. Analysis of the individual counts gave a standard deviation offour to ive percent for the stick method of this invention and 11.5percent for the standard plate count method.

Although this invention has been described with relation to the twoembodiments of the charge stick shown in the drawing, it will beunderstood by those skilled in the art that the shape of the stick andthe length, diameter and spacing of the holes may be varied to optimumarrangements suitable for either visual or automatic scanning. Othermodications will-readily appear to those skilled in the art and thisinvention should be limited only by the scope of the appended claims.

I claim:

l. A device for determining the concentration of microorganisms in aliquid which consists of a slab-like form provided with a plurality ofrelatively uniform openings therethrough; said openings being of acapillary size for holding liquid samples therein by capillaryattraction; said slab-like form being made of polymerized tetra-fluoroethylene.

2. A method for determining the concentration of microorganisms in -aliquid which consists in immersing a slab-like form of polymerizedtetra-fluoro ethylene having a plurality of capillary size aperturestherein into the liquid withdrawing said slab with its apertures lledwith the liquid and its surface adjacent said apertures void of saidliquid, subjecting it to incubating conditions in order to develop anyprogeny in the liquid filled apertures and determining the number ofprogeny so developed.

References Cited in the tile of this patent UNITED STATES PATENTS Lovells- May 4, 1954

1. A DEVICE FOR DETERMINING THE CONCENTRATION OF MICROORGANISMS IN ALIQUID WHICH CONSISTS OF A SLAB-LIKE FORM PROVIDED WITH A PLURALITY OFRELATIVELY UNIFORM OPENINGS THERETHROUGH; SAID OPENINGS BEING OF ACAPILLARY SIZE FOR HOLDING LIQUID SAMPLES THEREIN BY CAPILLARYATTRACTION: SAID SLAB-LIKE FORM BEING MADE OF POLYMERIZED TETRA-FLUOROETHYLENE.